It has been determined by gel filtration under denaturing conditions (70% formic acid) that the 26,000-dalton tryptic peptide of myosin light chain kinase contains one of the two sites of phosphorylation by cAMP-dependent protein kinase. A tryptic peptide containing the second site of phosphorylation appears to be associated with the 26,000-dalton peptide and to co-elute with it on gel filtration under native conditions (0.1M NH4HC03, 0.2M NaCl, pH 7.5). This second site is the one which cannot be phosphorylated when calmodulin is bound to myosin kinase and which exerts a regulatory effect on calmodulin binding and myosin light chain kinase activity. Present studies are under way to sequence the phosphorylated peptides of diphosphorylated myosin light chain kinase. One of these peptides will confirm a sequence previously determined and the second will be the site whose phosphorylation alters the ability of myosin kinase to bind calmodulin. An enzyme (MW 150,000) that methylates DNA at CpG sequences has been partially purified by Dr. Timothy Bestor. The enzyme preparation contains endogenous kinase activity which can phosphorylate the methylase. The methylase can also be a substrate for cyclic AMP-dependent protein kinase or for protein kinase C but only low levels of phosphate can be incorporated. There is no measurable change in methylase activity versus an unmethylated DNA substrate. Neither myosin light chain kinase nor a tyrosine kinase prepared from Rous sarcoma virus-induced rat tumors can phosphorylate the methylase. Current work is directed towards measuring the effect of dephosphorylation of the methylase by nonspecific phosphatases (alkaline phosphatase and a phosphatase purified from turkey gizzard smooth muscle) on enzyme activity.